We will attempt to use our unnatural amino acid mutagenesis methodology to genetically encode libraries of cyclic and linear peptides containing a diverse array of unnatural amino acids. These peptide libraries will be generated both intracellularly in bacteria and in a phage-displayed format to allow in vivo selections and display-based affinity panning, respectively. This strategy will enable direct genetic encoding of molecular structure, random or directed mutagenesis, selection, and biological amplification, while at the same time allowing chemical diversity beyond what the canonical 20 amino acid repertoire can offer. The integration of unnatural amino acids into genetically encoded peptide libraries should facilitate both evolutionary experiments and the generation of peptides with novel biological activities that may ultimately find therapeutic use. This will be achieved through the following specific aims: 1. The preparation and incorporation of novel unnatural amino acids with unique reactivities and with functional groups capable of targeting specific proteins involved in disease states (e.g., serine proteases, metalloproteases, etc.) 2. The use of intein-based intracellular cyclic peptide synthesis methodologies together with unnatural amino acid technology to generate novel cyclic peptide libraries and select specific enzyme inhibitors 3. The use of phage display systems to generate and screen libraries of peptides containing unnatural amino acids for inhibitors of specific protein targets. PUBLIC HEALTH RELEVANCE: The ability to employ unnatural amino acids in biological selections and screens of peptide libraries should complement current synthetic and biological methods for generating biologically active peptides. Importantly, it will allow us to combine the power of templated biological synthesis, mutation, amplification, and selection with the chemist's ability to synthesize amino acids with defined steric and electronic properties not found in the canonical amino acids. The demonstration that these libraries can be efficiently selected or screened to identify specific inhibitors of protein function may ultimately provide a new approach to the development of peptide therapeutics.